Characterization And Expression Of Genes Involved In The Abscisic Acid Biosynthesis And Metabolism During Ripening Of Mulberry Fruit

Mulberry tree(Morus alba L.) is a perennial woody plant which leaf is the main forage for silkworm(Bombyx mori L.) and the fruit was valued for it’s rich nutrient and flavor contents. Furthermore, mulberry fruit was popular with people for its unique taste. However, its value is constrained by its brief period of maturity.Previous study have proved that ABA was an important regulator during fruit ripening, participate in the regulation of pigment and sugar accumulation as well as fruit softening and absciss. However, the underlying mechanism of ABA plays during fruit ripening is still unknown. In this study, a new Morus cultivated of M. atropurpurea cv. Jialing No.40 fruit were used as the materials. The sequences of ABA biosynthesis related genes were obtained from Morus notabilis genome database and its deduced amino acid sequences were analyzed by using bioinformatics tools. We also using physiological biochemical and molecular biology method to study the mechanism of ABA biosynthesis related genes plays during fruit ripening. We hope this work could lay a foundation for further studying the role of ABA on mulberry(Morus alba L.) fruit maturity and senescence. Meanwhile, the expression pattern of NCEDs genes were analysised uder stress condition as well as ABA, fluridone, and sodium tungstate treatment in mulberry seedling. The main results we had obtained were shown as follows:1. Identification and bioinformatics analysis of the genes involved in ABA biosynthesis form mulberryIn this study, the sequences of six putative ABA biosynthesis related genes were obtained from Morus notabilis genome database, including one AAO gene, two ZEP genes and three NCED genes, and its deduced amino acid sequences were analyzed by using bioinformatics tools.Multiple-sequence alignment results showed that Mn NCEDs amino acids are relatively conserved in higher plants. Cluster analysis revealed that Mn NCED1-3 is closely related with dicotyledons plants and farther with monocotyledons plants. Transcription analysis indicated that the expression levels of ABA biosynthesis related genes were higher in leaves and the lowest expression was detected in roots. Particularly, the expression levels of Mn NCED2 and Mn NCED3 was higher in different plant tissues compared to other genes.Six ABA metabolism related genes which encoded 8’- hydroxylase were obtained from Morus notabilis genome database, belonging to CYP707 A gene family. The transcription expression results showed that all these genes had been detected an expression in five tissues except Mn CYP707A4 in male flower and Mn CYP707A6 in root and winter bud2. The change of ABA content during mulberry fruit ripeningWith the development of mulberry fruit, contents of ABA appeared a weak peak at the eighth day(small green), than increased rapidly from fourteenth day after full bloom with the pigments accumulation, weight increased, and fruit softening. At 37DAFB(full red), the contents of ABA reached the highest peak and the fruit fully maturity.3. Effects of exogenous ABA, glucose, sucrose, and fluridone on the abscission and maturation of Morus fruitFruits at degreen stage(26 DAF) were treated with ABA, and fluridone, after seven days statistics of the fruit abscission rate, results showed that ABA promote fruit abscisic while a delay of fruit ripening were found in fluridone treatment. Furthermore, the percentage of fruits at different stages of maturity was counted after ABA, glucose, sucrose, and fluridone treatment for 3d. The result indicated that the percentage of Morus fruits at full maturity following the sucrose treatment was higher than that following the control treatment, while the percentage of fruits at full maturity following the fluridone treatment did not obviously differ from that of the control. The results showed that sucrose was the factor that most significantly promoted ripening, while fluridone remarkably inhibited fruit ripening. The percentage of Morus fruits at full maturity following the ABA, sucrose, and glucose treatment was higher than that following the control treatment, while the percentage of fruits at full maturity following the fluridone treatment was decreased compared with control.4. Expression of ABA biosynthesis related genes in Morus during fruit development and ripeningThe expression of ABA biosynthesis related genes showed different patterns during fruit development and ripening. Among these genes, Mn NCED2 was expressed at relatively high levels and Mn ZEP1 and Mn ZEP2 were expressed at low levels, while Mn NCED1, Mn NCED3, and Mn AAO were expressed at lowest levels throughout the entire process of fruit development and ripening. The expression abundance of Mn NCED2 and Mn NCED3 were higher in the middle and latter periods than the early stage. However, the expression level of Mn NCED1 reached a peak at 21 DAFB and then decreased gradually until 37 DAFB. Correlation analysis of ABA content and relative gene transcriptional level were performed using SPSS Statistics for Windows, results showed that the expression of Mn NCED2 and Mn NCED3 have high Correlation with ABA content and may acted as a key role in ABA biosynthesis.The transcriptional expression levels of Mn NCED1, Mn AAO, and Mn ZEP1 was up-regulated at the fourth day after ABA treatment, and Mn NCED3 was up-regulated from the three day, while Mn ZEP2 always showed raised after the detached mulberry fruits were treated with ABA. The expression levels of Mn NCED1 and Mn ZEP2 showed down-regulated in the first and the three days after fluridone treatment, while Mn AAO and Mn ZEP1 increased in the fourth day, and Mn NECE1 was only down-regulated in the first days. However, the expression level of Mn NCED2 was down-regulated after both ABA and fluridone treatments.During fruit ripening, the expression of Ma CYP707A1, Ma CYP707A2, Ma CYP707A3 and Ma CYP707A4 were higher at pre-maturation stage and kept at a lower level during middle and post-maturation stages, which were opposite to the change of ABA content. These results suggested that Ma CYP707A1, Ma CYP707A2, Ma CYP707A3 and Ma CYP707A4 may be the key gene involved in ABA metabolism and plays an important role in regulating of ABA level.5. Effects of ABA, fluridone, tungstate sodium, and mannitol treatment on mulberry seeding growth and expression of Mn NCEDsThe main length of mulberry seeding stem and root were shortened significancely after ABA, mannitol, tungstate sodium, and fluridone treatment compared with control. However, the ratio of root and stem were improved by ABA and mannitol while decreased by fluridone and tungstate sodium, and the most significant decreased was found after tungstate sodium treatment. The expression of Mn NCED2 and Mn NCED3 in stem, root, and leaf were down-regulated by ABA treatment. However, expression of Mn NCED2 was upregulated by mannitol. After tungstate sodium treatment, expression levels of Mn NCED2 in stem and leaf and Mn NCED3 in all tissues were inhibited. Furthermore, expression level of Mn NCED3 was decreased by fluridone.